Hematopoietic stem cells (HSCs) generate all blood lineages throughout the life of an organism and are clinically important as the therapeutic agents of bone marrow transplants used to treat hematological disorders. However, the availability of transplant therapy is limited by difficulty in finding immunologically compatible donors. The ability to generate large numbers of autologous HSCs in vitro from induced pluripotent stem cells (iPSCs) is a major biomedical objective and has the potential to eliminate problems of cell availability. To date, it has not been possible to generate bona fide HSCs with high engraftment potential and multi-lineage reconstitution. One possible means of generating HSCs from iPSCs would be to reconstitute the full set of HSC specification signals in vitro, however identifying these signals requires further interrogation of the molecular mechanisms controlling HSC specification in vivo. In vertebrate embryos, HSCs arise from hemogenic endothelium (HE) lining the ventral floor of the embryonic dorsal aorta (DA). My work using the zebrafish model of developmental hematopoiesis has shown that trunk neural crest (NC) cells provide required inductive cues for HSC specification to the HE. To identify novel NC derived regulators of HSC specification with the goal of informing efforts to generate HSCs in vitro, I conducted a transcriptional profiling screen of trunk NC and endothelial cells. Using NC specific targeted deletion of genes in zebrafish embryos, I will determine the molecular mechanisms by which candidates identified in the screen instruct HSC specification. Additionally, I have identified the HE expressed G-protein coupled receptor Gpr182 and putative co-receptor Ramp2, as novel regulators of HSC specification. Importantly, knockdown of either gpr182 or ramp2 directly phenocopy the effect of NC ablation on HSC specification, suggesting that Gpr182 could mediating a NC derived HSC specification signal. Using targeted knock out and proteomics, I will characterize Gpr182 and Ramp2 in the control of HSC specification. Derivatives of trunk NC, specifically sympathetic neurons and mesenchymal stem cells, control mobilization of adult HSCs. The requirement for NC derivatives in HSC specification suggests a lifelong signaling relationship between these cell types. I will employ permanent lineage tracing approaches to determine if HSC specification and the control adult HSC mobilization require the same types of NC derivatives. The mentored phase of this proposal will occur at St. Jude Children?s Research Hospital, under Dr. Wilson Clements and will confirm candidate regulators of HSC specification and focus on generation of mutant/transgenic zebrafish lines for the independent phase. The independent phase will elucidate the molecular mechanisms by which novel regulators instruct HSC specification and on permanent lineage tracing of NC derivatives. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.
Hematopoietic stem cells (HSCs) are the self-renewing population of cells that generate all the blood lineages throughout the life of an organism and are biomedically important as the basic units of the bone marrow transplants used to treat a myriad of malignant and non-malignant hematological diseases. This proposal aims to characterize and mechanistically describe how trunk neural crest derived signals direct the initial specification of HSCs in the vertebrate embryo. In the long term, characterizing these signals will inform improvements to protocols aimed at generating HSCs in vitro for biomedical applications.
